I was a postdoctoral scientist in Prof. Taylor's laboratory from 1998 to 2001. My project was to determine the structure of the inhibited conformation of smooth muscle HMM and myosin. I used electron crystallography of 2-D arrays of dephosphorylated smooth muscle HMM. The protein was expressed by Dr. Kathy Trybus and the crystallography was done using frozen hydrated specimens. This is a unique capability of Prof. Taylor’s laboratory. The 3-D reconstructions showed an unusual interaction between the two myosin heads that explained most of the biochemistry of the inhibited state of this myosin. The result obtained with the HMM fragment was later confirmed using full length smooth muscle myosin and later by work done in the laboratory of Dr. Roger Craig in tarantula myosin filaments.
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Figure
Legend: Averaged projection of smooth muscle heavy meromyosin in
the dephosphorolated state preserved frozen hydrated in amorphous ice.
2D array was grown on a lipid monolayer.
Smooth muscle heavy meromyosin (HMM) is a truncated double-headed myosin molecule. Two-dimensional crystals bound to a lipid monolayer were examined by cryo-electron microscopy on a 300keV FEG microscope. Data was collected for untilted and tilted specimen and a 2D projection map was calculated giving a resolution of 2.8 nm. The crystals show unit cell dimensions of 12.9 by 28.1 nm suggesting that there are 2 HMM molecules per unit cell with P2 symmetry. |